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Special Issue  |   August 2020
Predicting Thioflavin Fluorescence of Retinal Amyloid Deposits Associated With Alzheimer's Disease from Their Polarimetric Properties
Author Affiliations & Notes
  • Yunyi Qiu
    Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada
  • Tao Jin
    Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada
  • Erik Mason
    Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada
  • Melanie C. W. Campbell
    Department of Physics and Astronomy, School of Optometry and Vision Science, Department of Systems Design Engineering, Centre for Bioengineering and Biotechnology, Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, Canada. Centre for Eye and Vision Research, Hong Kong
    Centre for Eye and Vision Research, Hong Kong
  • Correspondence: Melanie C.W. Campbell, Department of Physics and Astronomy, 200 University Ave W, Waterloo, ON, N2L 3G1, Canada. e-mail: mcampbel@uwaterloo.ca 
Translational Vision Science & Technology August 2020, Vol.9, 47. doi:https://doi.org/10.1167/tvst.9.2.47
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      Yunyi Qiu, Tao Jin, Erik Mason, Melanie C. W. Campbell; Predicting Thioflavin Fluorescence of Retinal Amyloid Deposits Associated With Alzheimer's Disease from Their Polarimetric Properties. Trans. Vis. Sci. Tech. 2020;9(2):47. doi: https://doi.org/10.1167/tvst.9.2.47.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To use machine learning in those with brain amyloid to predict thioflavin fluorescence (indicative of amyloid) of retinal deposits from their interactions with polarized light.

Methods: We imaged 933 retinal deposits in 28 subjects with post mortem evidence of brain amyloid using thioflavin fluorescence and polarization sensitive microscopy. Means and standard deviations of 14 polarimetric properties were input to machine learning algorithms. Two oversampling strategies were applied to overcome data imbalance. Three machine learning algorithms: linear discriminant analysis, supporting vector machine, and random forest (RF) were trained to predict thioflavin positive deposits. For each method; accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve were computed.

Results: For the polarimetric positive deposits, using 1 oversampling method, RF had the highest area under the receiver operating characteristic curve (0.986), which was not different from that with the second oversampling method. RF had 95% accuracy, 94% sensitivity, and 97% specificity. After including deposits with no polarimetric signals, polarimetry correctly predicted 93% of thioflavin positive deposits. Linear retardance and linear anisotropy were the dominant polarimetric properties in RF with 1 oversampling method, and no polarimetric properties were dominant in the second method.

Conclusions: Thioflavin positivity of retinal amyloid deposits can be predicted from their images in polarized light. Polarimetry is a promising dye-free method of detecting amyloid deposits in ex vivo retinal tissue. Further testing is required for translation to live eye imaging.

Translational Relevance: This dye-free method distinguishes retinal amyloid deposits, a promising biomarker of Alzheimer's disease, in human retinas imaged with polarimetry.

Introduction
Alzheimer's disease (AD) is a neurodegenerative disease that leads to cognitive impairment and, ultimately, death. AD is the primary cause of dementia. In 2010, the worldwide prevalence of dementia was 35.6 million and this number is predicted to triple by 2050.1 Currently, this disease can only be definitively diagnosed post mortem through the severity of 2 hallmarks: amyloid plaques (composed of misfolded amyloid-β protein) and neurofibrillary tangles (composed of tau protein), both of which begin to accumulate in the brain before symptoms of cognitive impairment.2 Detection of amyloid, tau, and neurodegeneration in the brain is recommended for detecting changes associated with the AD disease process in clinical trials of novel treatments.3 Confirmation of amyloid in the brain is currently achieved using positron emission tomography (PET) scans4 or analyzing the cerebral spinal fluid.5 However, both methods are invasive: PET scans use a radiative isotope injection,4 and a cerebral spinal fluid test requires extraction of fluid from the patient's spine.5 Currently, there is no effective cure for AD. However, it has been suggested that earlier detection of brain amyloid in prodromal disease could enable more successful treatment before damage from amyloid and tau accummulates.6 Furthermore, the amyloid, tau, and neurodegeneration framework allows for the incorporation of other biomarkers of amyloid in the brain.5 Therefore, it is important to develop a noninvasive method to detect amyloid in association with AD before AD diagnosis. Potential peripheral biomarkers of amyloid in the brain include detection of amyloid in the retina.7 
Many changes have been measured in the retina in association with AD.8 An accumulation of amyloid deposits has been found in the retinas of AD patients by our group (M.C.W. Campbell, et al. IOVS 2010;51:ARVO E-Abstract 5778) and others,6,9 including immunohistochemistry positivity for the presence of amyloid-β1012 (Yuchun Tsai, et al. IOVS 2014;55:ARVO E-abstract 523-524). We demonstrated that it is possible to detect amyloid deposits in the retina using polarized light in an imaging device with animal models (Michael Tokiyoshi Hamel, et al. IOVS 2016;57:ARVO E-Abstract 2216) and humans (Tao Jin, et al. IOVS 2017;58:ARVO E-Abstract 3367; David DeVries, et al. IOVS 2015;56:ARVO E-Abstract 2385), because the interactions of the deposits with polarized light are significantly different from those of the surrounding retina. Retinal deposits were confirmed to be amyloid via Thioflavin-S staining.13 Koronyo et al.14 have also demonstrated the feasibility of the in vivo detection of retinal amyloid deposits using curcumin staining. This method, however, requires the ingestion of curcumin for 2 to 10 days to bind with the deposits.14 Our group has also shown that the number of amyloid deposits in the anterior retina predicts the severity of AD pathology in the brain, including the severity of brain amyloid15 (Frank Corapi, et al. IOVS 2018;59:ARVO E-Abstract 1582). 
Thioflavin is a fluorescent marker of amyloid, including amyloid-β, known to occur in AD. Thioflavin also stains other amyloids,16 some of which are associated with other neurodegenerative diseases.17 In the amyloid, tau, and neurodegeneration diagnostic framework for AD, amyloid PET scans are an approved method of measuring brain amyloid.3 Similarly, a dye used in amyloid PET scans, Pittsburgh compound B, is an uncharged analog derived from thioflavin and thus marks amyloid and may not be specific to amyloid-β.18 
A number of studies have applied machine learning techniques to AD diagnosis. Two studies used a supporting vector machine (SVM) algorithm to distinguish those with AD from controls using neuroimaging data from magnetic resonance imaging.19,20 Another used random forest (RF) analysis on magnetic resonance imaging data to separate those with AD from healthy controls and compared the performance of RF with SVM.21 In optical coherence tomography, a SVM-based 3-dimensional segmentation of retinal layers has been reported in both diseased and normal retina.22 In addition, images taken with polarized light of morphologically similar algae have been classified with a convolutional neural network.23 
In those with brain amyloid, imaging the retina with polarized light (polarimetry) detects the majority of the deposits stained with Thioflavin-S (true positives), some deposits that are not stained by Thioflavin-S (false positives) and fails to detect a few Thioflavin-S–positive deposits (false negatives). The purpose of this study was to differentiate Thioflavin-S-positive amyloid deposits (true positives) from those deposits without Thioflavin-S fluorescence (false positives), based on the polarimetric images of the deposits to identify retinal amyloid deposits without the use of dye. To this end, we implemented and compared machine learning approaches, which can predict the presence of retinal amyloid fluorescence using only the information from images obtained with polarized light. 
Methods
Sample Preparation
Retinal Samples
Eyes and brains (N = 28) were obtained post mortem from donors in compliance with the Declaration of Helsinki. Informed consent was obtained from the donors and the research was approved by the Human Research Ethics Committee of the University of Waterloo. Upon post mortem examination (National Institute on Aging–Alzheimer's Institute guidelines2), 23 donors had a high cumulative score of AD neuropathologic change, 4 had a moderate score, and 1 had a low score; all had evidence of brain amyloid. Eyes were immersed in 10% formalin before dissecting, staining, and flat mounting the retina. Retinas were stained with 0.1% Thioflavin-S, counterstained with DAPI, and cover-slipped. Each retina was imaged with fluorescence and polarimetric microscopy to determine presumed amyloid deposits. In total, 920 polarization-positive retinal deposits found in anterior retinal layers from 28 individuals were analyzed, including 789 deposits with fluorescence signals and 131 deposits without fluorescence signals. An additional 16 deposits had only fluorescent signals; 13 of these deposits had no polarimetric signals and 3 had unclear polarimetric signals. 
Pure Amyloid Samples
Amyloid-β (Aβ) 1-42 was purchased from rPeptide (Athens, GA). One milliliter of 10 mM HEPES and 150 mM NaCl (pH of 7.4) was added to 0.5 mg of Aβ (1-42) and incubated at 37°C for 72 hours without shaking. Stained Aβ was prepared by adding 500 µL of filtered 1.25 mM Thioflavin-T in 50 mM of phosphate-buffered saline and allowing it to sit for 5 minutes before gently rinsing the amyloid deposited on glass with 50 µL of distilled water (×3). Samples were then blown dry with a gentle stream of compressed N2 and cover slipped. 
Mueller Matrix Polarimetry
An inverted transmission Nikon microscope was modified for polarimetric imaging to detect the interaction of retinal deposits with polarized light. A polarization state generator (PSG) and a polarization state analyzer (PSA) were placed before and after the sample, respectively. Both the PSG and PSA are composed of a linear polarizer and a quarter wave plate (QWP), with the polarizer followed by a QWP in the PSG and the opposite in the PSA. The light travels from the PSG through the sample to the PSA before being collected by the camera (Fig. 1). 
Figure 1.
 
A schematic diagram represents the setup of the microscope channel for polarimetric imaging. P1 and P2 are linear polarizers, \(\frac{\lambda }{4}\) marks quarter wave plates. The CCD is a camera with a charge-coupled device. PSA, polarization state analyzer; PSG, polarization state generator.
Figure 1.
 
A schematic diagram represents the setup of the microscope channel for polarimetric imaging. P1 and P2 are linear polarizers, \(\frac{\lambda }{4}\) marks quarter wave plates. The CCD is a camera with a charge-coupled device. PSA, polarization state analyzer; PSG, polarization state generator.
The QWP in the PSG is rotated to four angles (45°, 0°, −30°, and −60°) to generate four different input polarization states. The output at each setting was determined by rotating the QWP in the PSA to the same four angles. Thus, a combination of 16 measurements were performed for each sample. The images obtained were registered to compute the Mueller matrix of the sample at each image pixel. For a given pixel, the intensity, Ii recorded in the ith measurement is then,  
\begin{equation}{I_i} = {M_{PSA,i}}{M_s}{M_{PSG,i}}{S_{in}}\;\left( {i = 0,1,2, \ldots ,\;15} \right)\end{equation}
(1)
where Sin stands for the Stokes vector of the incident light and MPSA,i and MPSG,i are the Mueller matrices of the PSA and PSG, respectively. Because the Mueller matrices of these elements are known, the Mueller matrix at each pixel of the sample, Ms, can be determined and polarimetric properties calculated. 
Machine Learning Approach
Mean values and standard deviations of polarimetric properties (features) of the segmented deposits were used as feature inputs for machine learning algorithms which then learn to classify the deposits as members of the thioflavin positive or negative datasets labelled by their class. Thus, the algorithms are often referred to as classifiers. For definitions of machine learning terms please refer to Appendix A
Segmentation and Calculation of Properties of Polarimetric Images
From the 16-element Mueller matrix calculated at each pixel, the state of polarized light exiting from the sample for each possible state of light input can be calculated. The way in which the input polarized states are changed to the exiting states is described by the Mueller matrix of each pixel of the sample from which polarimetric properties are calculated. For example, one state of incident polarization could be preferentially absorbed (the property of diattenuation) or the refractive index seen by different polarizations could differ (the property of retardance) or polarized light could be changed to partially polarized light.24 
For each deposit, the means and standard deviations of 14 polarimetric properties were extracted from the Mueller matrix values of the segmented pixels and used as feature inputs for machine learning algorithms. Linear retardance (LR), circular retardance, and depolarization power were extracted by the polar decomposition method.25 Two polarimetric properties, linear anisotropy (LA) and circular anisotropy, were calculated from the Mueller matrix components.26 Four polarimetric properties computed by the Mueller matrix transformation technique were: metric A (a measure of LA), metric b (sensitive to small sources of scattered light in samples), metric t (related to the magnitude of the anisotropy) and metric x (related to the direction of the aligned fibrous structures).27,28 Linear and circular polarizance and diattenuation were directly obtained from the polarizance and diattenuation vectors of the Mueller matrix, respectively. The Q metric is a polarimetric property that contains information on depolarization, polarizance and diattenuation.29 Table 1 in Appendix B summarizes all 14 polarimetric properties with equations. Polarimetric properties of presumed amyloid deposits (plotted as images) were segmented from the surrounding retinal background by a custom polarimetry segmentation method (Erik Mason, et al. IOVS 2019;60:ARVO E-Abstract 179). 
Table 1.
 
ACC, SEN, and SPE of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 1.
 
ACC, SEN, and SPE of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Oversampling Methods for Solving Data Imbalance
As described in the Sample Preparation section, our 2 datasets do not have balanced deposit numbers (789 fluorescence positive and 131 fluorescence negative deposits). In this study, the minority (smaller) dataset contains samples with polarization signals but no fluorescence signals, and the majority (larger) dataset contains samples with both polarization and fluorescence signals. This imbalance, frequently observed in medical datasets, can lead to poor performance of most classification algorithms.30 Two oversampling methods were used to resolve data imbalance while maintaining the information of the original dataset: (1) sampling fluorescence negative retinal background and (2) the borderline-SMOTE algorithm.31 
Method 1: Retinal Oversampling—Adding the Surrounding Retinal Background
To supply more samples with no fluorescence signal, regions with the same shape as a polarimetric-positive deposit in a nearby fluorescence and polarimetric-negative retinal region were extracted (Fig. 2). The extracted region has no fluorescence signal and a weak polarization signal. We then calculated the means and standard deviations of the polarimetric signals from these regions and labeled them as nonfluorescence deposits. Of these regions, 658 were randomly selected from the eligible retina areas. As a result, we have an equal number (789) of fluorescence-positive and fluorescence-negative samples after oversampling. 
Figure 2.
 
An example of the retinal oversampling method. In the linear anisotropy image of a polarimetric-positive deposit, a region with the same shape (blue dashed line) as the deposit (red line) with no fluorescence signal is extracted as a fluorescence negative sample. Scale bar: 20 µm.
Figure 2.
 
An example of the retinal oversampling method. In the linear anisotropy image of a polarimetric-positive deposit, a region with the same shape (blue dashed line) as the deposit (red line) with no fluorescence signal is extracted as a fluorescence negative sample. Scale bar: 20 µm.
Method 2: Oversampling by Borderline-SMOTE
Another way of oversampling is to artificially generate data to add to the minority dataset. Here, an improved version of the SMOTE algorithm32—borderline-SMOTE31 was applied. This algorithm first performs a k-nearest neighbor analysis on the thioflavin negative (minority) dataset where the nearest neighbors may be within either or both minority and thioflavin positive (majority) datasets. Based on the type of nearest neighbors, deposits in the minority dataset are ranked into 3 subsets: noise, danger, and safe. Deposits in the noise subset have all nearest neighbors in the majority dataset; deposits in the safe subset have more than one-half of their nearest neighbors in the minority dataset. These subsets are relatively far from the border between the minority and majority datasets. Deposits in the danger subset have more than one-half but not all of their nearest neighbors in the majority dataset. New synthetic data are generated by performing the SMOTE algorithm only on the danger subset. 
Applying Machine Learning Algorithms
Because different polarimetric properties have different scales due to their nature (e.g., the LR ranges from 0° to 180°, whereas linear diattenuation ranges from 0 to 1), a widely used method called min-max scaling, which converts all polarimetric properties to have the same scale using formula (2) was applied to standardize polarimetric property scales.  
\begin{equation}{X_{scaled}} = \frac{{X - {X_{min}}}}{{{X_{max}} - {X_{min}}}}\end{equation}
(2)
where Xmin and Xmax represent the minimum and maximum theoretical values of a given polarimetric property. 
The rescaled combined thioflavin-negative and thioflavin-positive polarimetric datasets were then randomly split into 2 sets for training (80%) and testing (20%). During training, 10-fold cross-validation was performed to evaluate the accuracy (ACC) and robustness of the classifiers.33 Three different algorithms were trained to automatically determine the existence of a fluorescence signal: linear discriminant analysis (LDA), SVM, and RF. The hyperparameters (input settings, Appendix B, Table 2) of SVM and RF were optimized by the randomized search function provided in the scikit-learn library,34 which selects random combinations in a grid of hyperparameters to train the model and return the combination with the best ACC. 
Apart from the mean ACC returned from 10-fold cross-validation, we also evaluated the performance of each trained algorithm (classifier) by computing sensitivity (SEN), specificity (SPE), and area under the receiver operating characteristic curve (AUC) on the test set. The evaluations were repeated five times to establish standard deviations. A receiver operating characteristic curve was calculated for each classifier to compare their classification performance.35 
Finally, the variable importance36 was obtained from the RF analysis to assess the importance of each polarimetric property in predicting deposit fluorescence. The variable importance is calculated from the average decrease of the Gini impurity when each property is considered. At each node τ, the Gini impurity is calculated as,  
\begin{equation}Gini\left( {\rm{\tau }} \right) = 1 - \;\mathop \sum \nolimits_k {p_k}^2\end{equation}
(3)
where k stands for the class (or label) of each dataset, fluorescence positive or fluorescence negative; and Pk stands for the proportion of the sample placed in class k before and after splitting at a node. The property with the smallest Gini impurity is chosen for node splitting. 
Results
Visualization of Retinal Deposits
A Mueller matrix of a polarimetric and fluorescence positive deposit is shown in Appendix Figure A1. The deposit is clearly visible against the surrounding retina. 
Four examples of the polarimetric properties of retinal deposits (2 fluorescence positive and 2 fluorescence negative) are displayed in Figure 3. In general, these polarimetric properties change from pixel to pixel as well as between deposits and surrounding retina. These deposits have signals in polarimetric properties including LR and LA. Retinal deposits 1 and 2 and both pure protein (amyloid-β) deposits are positive for thioflavin fluorescence. The distributions of the strength of polarimetric properties across retinal deposits differ between fluorescence-positive and -negative deposits, but are similar between fluorescence-positive retinal and pure protein deposits. Using machine learning algorithms, our aim was to identify the differences in polarimetric properties between fluorescence-positive and -negative retinal deposits and predict the existence of a fluorescence signal. 
Figure 3.
 
Linear retardance (LR), linear anisotropy (LA), and fluorescence images of four retinal deposits (1 and 2 have fluorescence signals whereas 3 and 4 do not) and 2 pure Aβ-42 protein deposits with fluorescence signals. Range of LR: [0°, 180°], LA: [0, 1]. Scale bar in the upper left panel: 20 µm.
Figure 3.
 
Linear retardance (LR), linear anisotropy (LA), and fluorescence images of four retinal deposits (1 and 2 have fluorescence signals whereas 3 and 4 do not) and 2 pure Aβ-42 protein deposits with fluorescence signals. Range of LR: [0°, 180°], LA: [0, 1]. Scale bar in the upper left panel: 20 µm.
Initially, 16 deposits seemed to be fluorescent positive and polarization negative, but a comparison of the averages of polarization signals from each deposit and surrounding retina found that 3 deposits had weak polarization signals. None of the 16 deposits were included in the datasets. 
Performance of the Three Algorithms in Classification
The performance of the 3 algorithms (LDA, SVM, and RF) in classification was assessed using 3 different methods (without oversampling, retinal oversampling method, and oversampling by borderline-SMOTE) and the outcomes are shown in Table 1. The receiver operating characteristic curves are shown in Figure 4 and their AUCs are summarized in Table 2
Figure 4.
 
Receiver operating characteristic (ROC) curves for fluorescence signal prediction by the three classifiers under three sampling strategies. The true-positive rate is the sensitivity and the false-positive rate is 1 – specificity. LDA, linear discriminant analysis; RF, random forest; SVM, supporting vector machine.
Figure 4.
 
Receiver operating characteristic (ROC) curves for fluorescence signal prediction by the three classifiers under three sampling strategies. The true-positive rate is the sensitivity and the false-positive rate is 1 – specificity. LDA, linear discriminant analysis; RF, random forest; SVM, supporting vector machine.
ACC is the mean accuracy from 10-fold cross-validation. Shown are the mean values with standard deviations obtained from shuffling and recalculating the training and test sets five times. From Table 1, the SPEs of all classifiers are significantly improved after applying oversampling methods. Among the three classifiers, the performance of LDA is the most sensitive to the choice of oversampling method, with ACC, SEN and SPE decreasing by 11% on average in the borderline-SMOTE method compared with retinal oversampling. SVM had a lower SEN, but a higher SPE in borderline-SMOTE oversampling, giving a negligible change in ACC. Both SVM and RF perform well and are relatively insensitive to the oversampling method with ACC of RF slightly above SVM at 94% to 95%. 
Overall, the AUCs of the three classifiers in Table 2 increased after using oversampling methods, from previously less than 90% to more than 95%, except for the LDA classifier in the borderline-SMOTE method, which was not different from before oversampling. The AUC values for the RF and SVM classifiers were not significantly different between the 2 oversampling methods. The RF classifier has the highest AUC in comparison to LDA and SVM using the borderline-SMOTE oversampling (AUC = 0.986). 
Importance of Polarimetric Properties
We ranked the polarimetric properties by their importance as feature inputs in RF analysis in Figure 5 for retinal oversampling and in Figure 6 for the borderline-SMOTE oversampling method. 
Figure 5.
 
The variable importance of 28 polarimetric properties as feature inputs (means and standard deviations [Std] of 14 polarimetric properties) from a random forest analysis with retinal oversampling. The sum of the variable importance of all features is 1.
Figure 5.
 
The variable importance of 28 polarimetric properties as feature inputs (means and standard deviations [Std] of 14 polarimetric properties) from a random forest analysis with retinal oversampling. The sum of the variable importance of all features is 1.
Figure 6.
 
The variable importance of 28 feature inputs (means and standard deviations [Std] of 14 polarimetric properties) for a random forest analysis with borderline-SMOTE oversampling. The sum of the variable importance of all feature is 1.
Figure 6.
 
The variable importance of 28 feature inputs (means and standard deviations [Std] of 14 polarimetric properties) for a random forest analysis with borderline-SMOTE oversampling. The sum of the variable importance of all feature is 1.
In retinal oversampling (Fig. 5), LR and LA were the two dominant polarimetric properties (features) in predicting Thioflavin-S fluorescence. Their means and standard deviations together accounted for 74.8% of the total variable importance. Therefore, other feature inputs with lower importance were excluded, and the analysis was rerun with only mean and standard deviation of these two polarimetric properties as feature inputs. Table 3 summarizes the results of retinal oversampling before and after this selection. 
Table 2.
 
Summary of AUC of the ROCs of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 2.
 
Summary of AUC of the ROCs of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 3.
 
Classification Performance by Retinal Oversampling Before and After Applying Feature Selection
Table 3.
 
Classification Performance by Retinal Oversampling Before and After Applying Feature Selection
From Table 3, there was a decrease in the ACC and SEN for LDA and a small decrease in ACC for RF, but no other significant changes after performing feature selection. Although only four polarimetric properties were used as features, SVM and RF classifiers still achieved more than 90% in ACC. Thus, other polarization metrics can be excluded to speed up the analysis with little loss in performance in predicting deposit fluorescence. 
In contrast, the variable importance distribution of each polarization feature was much different under borderline-SMOTE oversampling in Figure 6 than in retinal oversampling with a much smaller range of contribution values (contributions range from 1.5% to 10.1%). Therefore, feature selection was not applied for the borderline-SMOTE oversampling method because there was no evidence of dominant polarimetric properties. 
Discussion
There are some limitations in this study. First, it is difficult to quantitatively evaluate the similarity between an oversampled dataset and the actual dataset. This factor may affect the generalization and robustness of our models. Because retinal deposits are found sparsely distributed across the retina, retinal oversampling is justified. Although more data from retinal deposits with polarimetric but no fluorescence signals would further validate the reliability of the oversampling methods, because polarimetry predicts thioflavin positivity with high ACC, such false-negative deposits are scarce. 
Second, the limited number of retinas (n = 28) and deposits (n = 920, before oversampling) involved in this study could bias our models. Thus, it would be helpful to analyze more retinas to enhance the generalization of the models before moving to a clinical setting. However, the number of paired retinas and brains available post mortem is limited. In vivo studies could compare retinal polarimetry and in vivo brain amyloid measurements in larger sample sizes. 
Third, other classification models such as convolutional neural networks should be tested to see if they can further improve the already excellent ACC of the classification of thioflavin positive (presumed amyloid) versus thioflavin-negative deposits. Considering the ACC as well as the computation/memory and time costs of the models, we could then select the one that would be best for clinical application. 
Amyloid stained with Congo red is known to interact with polarized light producing an apple green birefringence signal.37,38 We have shown that unstained pure amyloid-β and thioflavin positive retinal amyloid deposits also interact with polarized light and show very similar variations of LR and depolarization across deposits (Corapi F, et al. IOVS 2018;59:ARVO E-Abstract 1582). These similarities and thioflavin positivity39 are presumably due to the fibrillary, well-ordered structure of these deposits.39 Thus, it is not unexpected that LR and polarimetric properties which are related to LR (such as LA) are important to distinguishing thioflavin positive (amyloid) deposits from those that are not thioflavin positive. 
Amyloid deposits show a range of polarimetric property strengths, both within and between deposits, potentially owing to a variety of factors, including variations in thickness (thinner regions have weaker signals, usually at the edge of deposits (e.g., deposit 2 in Fig. 3), in the uniformity of orientation of the fibrils and potential differences in amyloid species. Thioflavin has been known to stain amyloid species other than amyloid-β, such as alpha-synuclein.16 By choosing retinas from donors with a brain pathology of amyloid deposits of the Alzheimer's type, we expect the majority of thioflavin positive deposits to contain amyloid-β. The lack of polarization signals for a small number (n = 16) of thioflavin positive deposits may indicate one or more of the following: thinner deposits (Tao Jin, et al. IOVS 2017;58:ARVO E-Abstract 3367), more disordered deposits, or the presence of other impurities (analogous to neuritic material found in brain plaques).40 
Despite the range of polarimetric properties of deposits in the retina, which indicates differing interactions with polarized light, in the present study, we trained machine learning classifiers to precisely and robustly distinguish polarimetry-positive retinal deposits with and without thioflavin fluorescence signals. In turn, thioflavin positivity defines a presumed amyloid deposit. In this study, the RF and SVM classifiers exhibited high ACC, SEN, and SPE. Two oversampling methods proved to be useful for dealing with the larger numbers of fluorescence positive versus negative deposits and improved the SPE, AUC and, in all but one case, the ACC of the classifications. 
Using the RF algorithm with the retinal oversampling method, we found that the means and standard deviations of two polarimetric properties—LR and LA—have superior performance in predicting fluorescence positivity. We can use only these four properties as feature inputs to produce excellent classification performance. From the perspective of the physical meanings of these two polarimetric properties, LR is directly related to the linear birefringence which results from the ordered alignment of the fibrils. LA is a measure of the polarimetric anisotropy of samples that can be interpreted as the different optical responses to light polarized in different directions caused by the samples’ LR and linear diattenuation.26,41 LA is postulated to have a periodic variation that reflects the geometric structure of samples,26 such as the orientation of fibrillary structures. The high importance of these two polarimetric properties suggests that the ordered arrangement of fibrils may be the key to distinguishing between fluorescent and nonfluorescent deposits. Given the similarities between LR and LA patterns in deposits in Figure 3, for amyloid, retardation seems to contribute more to LA than diattenuation. 
In the case of the second (borderline-SMOTE) oversampling method, the variable importance returned from RF analysis indicates that LA and LR are no longer dominant variables. The contributions of all variables are within one order of magnitude. This difference in variable contribution is likely caused by the differing data introduced by the two oversampling methods. We performed a t-distributed stochastic neighbor embedding (t-SNE)42 (see Appendix A) to show the distributions of the polarimetric properties of each deposit for each oversampling method. The t-SNE algorithm maps the local distance information from the original high-dimensional space (of 14 properties) to a low-dimensional space (two properties). Perplexity, whose value is varied, provides a smooth measurement of the number considered as neighbors, which alters the algorithm's attention on local and global information (local variations dominate at a small perplexity). The t-SNE visualization plots for differing values of perplexity are displayed in Figure 7a for the retina oversampling method and Figure 7b for the borderline-SMOTE oversampling method. 
Figure 7.
 
Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of the polarization properties of the fluorescent positive and fluorescent negative datasets from (a) the retinal oversampling method and (b) the borderline-SMOTE oversampling method under different values of perplexity. The distribution focus moves from the local to the global as the perplexity increases. FP and FN are fluorescent positive and negative deposits, respectively. T_SNE_1 and T_SNE_2 are the axis of the 2-dimensional space to which the t-SNE mapped the higher dimensions.
Figure 7.
 
Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of the polarization properties of the fluorescent positive and fluorescent negative datasets from (a) the retinal oversampling method and (b) the borderline-SMOTE oversampling method under different values of perplexity. The distribution focus moves from the local to the global as the perplexity increases. FP and FN are fluorescent positive and negative deposits, respectively. T_SNE_1 and T_SNE_2 are the axis of the 2-dimensional space to which the t-SNE mapped the higher dimensions.
We can see in the t-SNE perplexity plots (Figs. 7a and 7b), that the data points of fluorescent negative and fluorescent positive areas are somewhat more separated in the retinal oversampling method than in the borderline-SMOTE oversampling method. As the perplexity increases, the sample distribution with negative fluorescence in retinal oversampling tends to aggregate at both ends of the dataset, whereas the borderline-SMOTE oversampling method produces a more complex data distribution. This finding implies that the properties of deposits identified in the borderline-SMOTE oversampling are closer to those of the fluorescent-positive deposits than in retinal oversampling. The differing data distributions are consistent with lower ACC for LDA with borderline-SMOTE. It is also not surprising that, for the two oversampling methods, differing polarimetric properties differentiate fluorescent positive from fluorescent-negative deposits. 
The methods described herein predict from the polarimetric properties of retinal deposits, with high ACC, the existence of amyloid with thioflavin positive fluorescence signals. We have shown that, in combination with machine learning algorithms, imaging using Mueller matrix polarimetry can detect amyloid positive deposits in the ex vivo retina without using a dye. We have previously reported that the polarimetric signals from retinal amyloid deposits measured in double pass, analogous to the live eye imaging method proposed in Campbell's patents,43 give polarimetric signals which are twice as large as those seen in single pass measurements (Photonics North presentation, 2016; M.C.W. Campbell, et al. IOVS 2020; 61:ARVO E-Abstract 1850). Our preferred live eye implementation uses a confocal scanning laser ophthalmoscope combined with polarimetry. Using the methods described here, we expect that thioflavin-positive retinal deposits could be identified with high ACC in live eye imaging. In turn, we have previously shown that the number of retinal amyloid deposits predicts the severity of amyloid in the brain.15 Therefore, in vivo dye-free polarimetric imaging of deposits in the retina could determine brain amyloid noninvasively, in contrast with current invasive methods. The results reported here are an important step towards the development of a novel, noninvasive, clinical method of imaging retinal amyloid as a predictor of brain amyloid. 
Acknowledgments
The authors thank Monika Kitor and Rachel Redekop for retinal image collection and manuscript editing. Second, we thank Nathan DeJong for editing Appendix A. Additionally, we acknowledge our collaborators at the University of British Columbia, Ian Mackenzie, Ging-Yuek Robin Hsiung and Veronica Hirsch for tissue samples and corresponding brain pathologies. Much of this work is included as part of Yunyi Qiu’s MSc thesis which has been submitted for examination to the University of Waterloo. 
Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research, Canada (CIHR). 
Disclosure: Y. Qiu, P; T. Jin, None; E. Mason, None; M.C.W. Campbell, LumeNeuro (I,S); University of Waterloo (P) 
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Appendix A: Definitions of Machine Learning Terms not Defined in the Text 
Words and phrases in bold are used in the main text. 
Feature: A feature is a property input to the machine learning algorithm. In this article we use polarimetric properties which describe interactions of the sample with polarized light. 
Figure A1.
 
A spatially resolved Mueller matrix (MM) of a polarimetric and fluorescence positive deposit. Elements are coded by row and column number (M00 to M33) and each pixel position has 16 elements associated with it. The matrix elements are normalized by M00. Scale bar: 20 µm.
Figure A1.
 
A spatially resolved Mueller matrix (MM) of a polarimetric and fluorescence positive deposit. Elements are coded by row and column number (M00 to M33) and each pixel position has 16 elements associated with it. The matrix elements are normalized by M00. Scale bar: 20 µm.
Datasets of each class: The groups into which the data is classified, each of which has a unique label, are referred to as classes. In our case, we have 2 datasets of deposits labelled with the classes, thioflavin positive and thioflavin negative, both members of which are described by polarimetric properties. When datasets contain unequal numbers of data, this is referred to as data imbalance
Machine learning algorithm: Computer programs (algorithms) that can “learn” from training data and improve their performance in predicting the class of input testing data. Our 2 datasets are labelled thioflavin positive and thioflavin negative. After learning training, these programs are given testing data, which they classify into the classes. These algorithms are also known as classifiers. The algorithms we test are: 
Linear discriminant analysis (LDA): Uses training data to learn linear equations that produce values that are close for members of the same dataset, and far apart for members of different datasets. These equations are then used to classify testing data into classes
Supporting vector machine (SVM): When training, SVM finds a decision boundary in the feature space to separate the different datasets as far as possible. Testing data are classified based on which side of the boundary they fall on. SVM heavily weights data close to the decision boundary. 
Random forest (RF): Uses many decision trees to generate a consensus decision. A decision tree classifies data points by using multiple properties to differentiate data into separate classes. When training, many trees fit a random subset of the training set. Each data point is available to be sampled multiple times during training. This is called bootstrapping. When testing, bootstrapping again generates many subsets, and each is classified. This classification uses a decision tree based on Gini impurity. Data points are then classified into the class where it was most frequently placed. 
Gini impurity: is a commonly used metric in RF decision trees to decide the optimal property to split the data into subsets. In general, as the splitting continues, the amount of data in branch nodes of a decision tree which belong to the same class should increase, this implies that the “purity” of the nodes is increasing. The purity of a dataset varies inversely with Gini impurity (Equation 3). The property with the smallest Gini impurity is chosen for node splitting. The decrease in Gini impurity at each node over the tree is summed to calculate the variable importance of each property
Oversampling strategy: If the datasets of each class are not balanced in their number of data points, these methods can be applied to increase the data points in the dataset with lower numbers (minority, in our case thioflavin negative deposits). Our majority dataset consists of thioflavin positive deposits. 
K-nearest neighbor analysis, used in oversampling: Determine the k points nearest a given point (from the nearest to the kth nearest). The points are defined by the values of the mean and standard deviation of each of the polarimetric properties. The nearest points have the most similar properties. 
SMOTE algorithm: A commonly used algorithm to generate synthetic samples (oversample) in the minority dataset to balance the number in the majority dataset. The algorithm begins by searching for the k-nearest neighbors of the same class (k is usually set to 5) for every sample in the minority dataset, then randomly generates a number of new synthetic data points along the line between the minority datapoint and its nearest neighbors. The number of synthetic data points matches the total number in the minority dataset to the majority dataset
Borderline-SMOTE oversampling: Based on SMOTE oversampling, borderline-SMOTE only performs the SMOTE algorithm on examples in the minority dataset that have nearest neighbors in the majority dataset as these are easily misclassified. 
Cross-validation: (10-fold): Divides the data into 10 subsets with similar size. One subset is used as the testing data and the other 9 are the training data. This is repeated for the other 9 subsets and the average of the 10 results is output. 
t- D istributed stochastic neighbor embedding (t-SNE) 42 test: A machine learning method for dimension reduction that can help to identify the patterns of properties in the datasets. The main advantage of t-SNE over other dimension reduction methods (such as principal component analysis) is the ability to maintain local patterns in the dataset. This means that similar points in high-dimensional space are still close to each other in a lower-dimensional projection. 
Perplexity is a parameter that controls the dimension reduction in t-SNE. It can be interpreted as the nearest neighbors considered when matching the original (high-dimensional space) and the fitted (low-dimensional space) of each point. A smaller perplexity means only a few points are considered as the nearest neighbors when doing fitting, which highlights the information local to the data points. A higher perplexity means that more points are considered as the nearest neighbors, giving a more “global view.” 
Appendix B: 
  
Table B1.
 
Equations for the Polarimetric Properties Used as Features in the Machine Learning Algorithms
Table B1.
 
Equations for the Polarimetric Properties Used as Features in the Machine Learning Algorithms
Table B2.
 
Hyperparameters of SVM and RF Where the Parameters Used Are Defined in34
Table B2.
 
Hyperparameters of SVM and RF Where the Parameters Used Are Defined in34
Figure 1.
 
A schematic diagram represents the setup of the microscope channel for polarimetric imaging. P1 and P2 are linear polarizers, \(\frac{\lambda }{4}\) marks quarter wave plates. The CCD is a camera with a charge-coupled device. PSA, polarization state analyzer; PSG, polarization state generator.
Figure 1.
 
A schematic diagram represents the setup of the microscope channel for polarimetric imaging. P1 and P2 are linear polarizers, \(\frac{\lambda }{4}\) marks quarter wave plates. The CCD is a camera with a charge-coupled device. PSA, polarization state analyzer; PSG, polarization state generator.
Figure 2.
 
An example of the retinal oversampling method. In the linear anisotropy image of a polarimetric-positive deposit, a region with the same shape (blue dashed line) as the deposit (red line) with no fluorescence signal is extracted as a fluorescence negative sample. Scale bar: 20 µm.
Figure 2.
 
An example of the retinal oversampling method. In the linear anisotropy image of a polarimetric-positive deposit, a region with the same shape (blue dashed line) as the deposit (red line) with no fluorescence signal is extracted as a fluorescence negative sample. Scale bar: 20 µm.
Figure 3.
 
Linear retardance (LR), linear anisotropy (LA), and fluorescence images of four retinal deposits (1 and 2 have fluorescence signals whereas 3 and 4 do not) and 2 pure Aβ-42 protein deposits with fluorescence signals. Range of LR: [0°, 180°], LA: [0, 1]. Scale bar in the upper left panel: 20 µm.
Figure 3.
 
Linear retardance (LR), linear anisotropy (LA), and fluorescence images of four retinal deposits (1 and 2 have fluorescence signals whereas 3 and 4 do not) and 2 pure Aβ-42 protein deposits with fluorescence signals. Range of LR: [0°, 180°], LA: [0, 1]. Scale bar in the upper left panel: 20 µm.
Figure 4.
 
Receiver operating characteristic (ROC) curves for fluorescence signal prediction by the three classifiers under three sampling strategies. The true-positive rate is the sensitivity and the false-positive rate is 1 – specificity. LDA, linear discriminant analysis; RF, random forest; SVM, supporting vector machine.
Figure 4.
 
Receiver operating characteristic (ROC) curves for fluorescence signal prediction by the three classifiers under three sampling strategies. The true-positive rate is the sensitivity and the false-positive rate is 1 – specificity. LDA, linear discriminant analysis; RF, random forest; SVM, supporting vector machine.
Figure 5.
 
The variable importance of 28 polarimetric properties as feature inputs (means and standard deviations [Std] of 14 polarimetric properties) from a random forest analysis with retinal oversampling. The sum of the variable importance of all features is 1.
Figure 5.
 
The variable importance of 28 polarimetric properties as feature inputs (means and standard deviations [Std] of 14 polarimetric properties) from a random forest analysis with retinal oversampling. The sum of the variable importance of all features is 1.
Figure 6.
 
The variable importance of 28 feature inputs (means and standard deviations [Std] of 14 polarimetric properties) for a random forest analysis with borderline-SMOTE oversampling. The sum of the variable importance of all feature is 1.
Figure 6.
 
The variable importance of 28 feature inputs (means and standard deviations [Std] of 14 polarimetric properties) for a random forest analysis with borderline-SMOTE oversampling. The sum of the variable importance of all feature is 1.
Figure 7.
 
Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of the polarization properties of the fluorescent positive and fluorescent negative datasets from (a) the retinal oversampling method and (b) the borderline-SMOTE oversampling method under different values of perplexity. The distribution focus moves from the local to the global as the perplexity increases. FP and FN are fluorescent positive and negative deposits, respectively. T_SNE_1 and T_SNE_2 are the axis of the 2-dimensional space to which the t-SNE mapped the higher dimensions.
Figure 7.
 
Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of the polarization properties of the fluorescent positive and fluorescent negative datasets from (a) the retinal oversampling method and (b) the borderline-SMOTE oversampling method under different values of perplexity. The distribution focus moves from the local to the global as the perplexity increases. FP and FN are fluorescent positive and negative deposits, respectively. T_SNE_1 and T_SNE_2 are the axis of the 2-dimensional space to which the t-SNE mapped the higher dimensions.
Figure A1.
 
A spatially resolved Mueller matrix (MM) of a polarimetric and fluorescence positive deposit. Elements are coded by row and column number (M00 to M33) and each pixel position has 16 elements associated with it. The matrix elements are normalized by M00. Scale bar: 20 µm.
Figure A1.
 
A spatially resolved Mueller matrix (MM) of a polarimetric and fluorescence positive deposit. Elements are coded by row and column number (M00 to M33) and each pixel position has 16 elements associated with it. The matrix elements are normalized by M00. Scale bar: 20 µm.
Table 1.
 
ACC, SEN, and SPE of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 1.
 
ACC, SEN, and SPE of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 2.
 
Summary of AUC of the ROCs of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 2.
 
Summary of AUC of the ROCs of the 3 Machine Learning Classifiers Under 3 Sampling Strategies
Table 3.
 
Classification Performance by Retinal Oversampling Before and After Applying Feature Selection
Table 3.
 
Classification Performance by Retinal Oversampling Before and After Applying Feature Selection
Table B1.
 
Equations for the Polarimetric Properties Used as Features in the Machine Learning Algorithms
Table B1.
 
Equations for the Polarimetric Properties Used as Features in the Machine Learning Algorithms
Table B2.
 
Hyperparameters of SVM and RF Where the Parameters Used Are Defined in34
Table B2.
 
Hyperparameters of SVM and RF Where the Parameters Used Are Defined in34
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